This invention relates to antibodies, methods for obtaining antibodies and assays for using such antibodies. More specifically, the invention relates to ungulate PrP antibodies methods of obtaining antibodies which specifically bind to naturally occurring forms of PrP from ungulates.
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not readily infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (xe2x80x9cPrPxe2x80x9d) (Bolton et al. (1982), Science 218:1309-11; Prusiner et al. (1982), Biochemistry 21:6942-50; McKinley et al.(1983), Cell 35:57-62). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene (Basler et al.(1986), Cell 46:417-28) and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrPC into a modified form called PrPSc.
It appears that PrPSc is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S. B.(1991), Science 252:1515-1522. The most common prion diseases of animals are scrapie of sheep and goats, and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith, J. and Wells (1991), Microbiol. Immunol. 172:21-38). Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) (Gajdusek, D. C., (1977) Science 197:943-960; Medori et al.(1992), N. Engl. J. Med. 326:444-449). The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
BSE is also a major socioeconomic problem, particularly in Britain. More than 175,000 cattle, primarily dairy cows, have died of BSE during the past decades. Tests conducted by the British government on cattle killed over a 30 month period suggest that about 0.3% of the 749,631 tested, or 2249 cattle may have had BSE even though they did not display any outward symptoms. On Mar. 27, 1996, the European Union (EU) placed a ban on the export of British bovine products, including: live bovine animals, their semen and embryos; meat of bovine animals slaughtered in UK; products obtained from bovine animals slaughtered in UK which are liable to enter the animal feed or human food chain; materials destined for use in medicinal products, cosmetics or pharmaceutical products; and mammalian derived meat and bone-meal. This ban has cost the British farming industry more than 1.5 billion pounds since it was imposed, and left many farmers bankrupt. Although the ban was lifted Aug. 1, 1999, both France and Germany still ban the import of British bovine products, contrary to the EU ruling.
The importance of detecting BSE has been heightened by the possibility that bovine prions have been transmitted to humans who developed new variant Creutzfeldt-Jakob disease (nvCJD) (G. Chazot et al.(1996), Lancet 347:1181; R. G. Will et al. (1996), Lancet 347:921-925). Earlier studies had shown that the N-terminus of PrPSc could be truncated without loss of scrapie infectivity (S. B. Prusiner et al.(1982), Biochemistry 21:6942-6950; S. B. Prusiner et al. (1984), Cell 38:127-134) and correspondingly, the truncation of the N-terminus of PrPSc still allowed its conversion into PrPSc (M. Rogers et al.(1993), Proc. Natl. Acad. Sci. USA 90:3182-3186). The ability of transmission of nvCJD from cattle to humans has been confirmed through in vivo testing, suggesting that the December 20 issue of Proceedings of National Academy of Sciences undermining the comforting presumption that the documented xe2x80x9cspecies barrierxe2x80x9d is relevant to this new strain (M. R. Scott et al. (1999), Proc. Natl. Acad. Sci. USA 96:15137-15142).
The presence of PrPSc in tissues of humans or animals is indicative of prion infection. PrPSc is the invariant component of prion infection and is the only disease-specific diagnostic marker that can be readily detected by immunoassay in the brains of clinically ill animals and humans Meyer et al. (1986), Proc. Natl. Acad. Sci. USA, 83:3693-7; Serban et al. (1990), Neurology, 40:110-117; Taraboulos et al. (1992), Proc. Natl. Acad. Sci. USA. 89:7620-7624; Grathwohl, K. U. D., M. Horiuchi et al. (1997), Virol. Methods 64:205-216. Unfortunately, PrPSc assays are positive only when the prion titer is high, while detection of low levels of PrPSc has been problematic. It has also proven difficult to measure low levels of PrPSc in the presence of high levels of PrPC.
Given the enormity of the potential effect of BSE on the world wide cattle population and the affected cattle population in Great Britain, there is a great need for a method of assessing bovine infection with BSE to protect the cattle populations. Given the potential health risk to the human population, more sensitive methods for detection of bovine prions are urgently needed.
The present invention provides antibodies that will specifically bind with a high degree of affinity to a native ungulate PrPC and/or a denatured ungulate PrPSc, but not to a native ungulate PrPSc. The antibodies are also highly specific, i.e. do not bind to other proteins. The antibodies are useful in numerous applications, and particularly for determining prion infection in ungulates. The antibodies are characterized by one or more of the following features (1) an ability to bind to native PrPC and denatured PrPSc, but not native PrPSc, with specificity, (2) an ability to bind to PrPC or denatured PrPSc in situ i.e., will only bind to PrPSc in a cell culture or in vivo if the prion protein has been treated (e.g. denatured). Preferred antibodies are further characterized by an ability to (4) bind to a PrPC protein of only a specific species of mammals e.g., bind to bovine PrPC and not to PrPC of other mammals.
An important object is to provide antibodies which bind to a native form of ungulate PrPC.
Another object is to provide antibodies which specifically bind to epitopes of PrPC of a specific species of animal (e.g. bovine PrPC) and not to the PrPC of other species of animals (e.g. mouse PrPC).
Still another object is to provide specific methodology to allow others to generate a wide range of specific antibodies characterized by their ability to bind one or more types of PrPC proteins from one or more species of ungulates.
Another object of the invention is to provide an assay for the detection of PrPSc in an ungulate using the antibodies of the invention.
An advantage of the invention is that it provides a fast, efficient cost effective assay for detecting the presence of PrPSc in an ungulate sample.
A specific advantage is that the assay can be used as a screen for the presence of prions (i.e., PrPSc) in products such as pharmaceuticals (derived from natural sources) food, cosmetics or any material which might contain such prions and thereby provide further assurances as to the safety of such products.
Another advantage is that the antibodies can be used with a compound which denatures PrPSc thereby providing for a means of differentiating levels of PrPC and PrPC+PrPSc in a sample.
A feature of the invention is that it uses phage display libraries in the creation of the antibodies.
Another feature of the invention is that the phage are genetically engineered to express a specific binding protein of an antibody on their surface.
An aspect of the invention is to provide a therapeutic antibody which prevents or treats prion disease in ungulates and specifically in cows.
Another aspect of the invention is to provide a means for certifying certain products as being prion free.
These and other aspects, objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the chimeric gene, assay method, and transgenic mouse as more fully described below.